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. 2022 Mar;12(3):e2464.
doi: 10.1002/brb3.2464. Epub 2022 Feb 1.

Involvement of the BDNF-TrkB-KCC2 pathway in neuropathic pain after brachial plexus avulsion

Shuo Zhao  1   2 , Fengyu Wang  1 , Li Wang  1 , Yali Xu  1 , Li Lv  1 , Wenxu Duan  1 , Runze Bai  1 , Zhao Meng  2 , Xinzhong Shao  1
Affiliations

Involvement of the BDNF-TrkB-KCC2 pathway in neuropathic pain after brachial plexus avulsion

Shuo Zhao et al. Brain Behav. 2022 Mar.

Abstract

Introduction: Brachial plexus avulsion significantly increased brain-derived neurotrophic factor (BDNF) release in the spinal cord. Here we investigated the involvement of the BDNF-TrkB-KCC2 pathway in neuropathic pain caused by BPA injury. We hypothesized that activation of BDNF-TrkB may inhibit neuronal excitability by downregulating KCC2 to maintain a high intracellular Cl-concentration. We established a neuropathic pain rat model by avulsion of the lower trunk brachial plexus, and investigated the effects of the TrkB-specific antibody K-252a on the expression of BDNF, TrkB, and KCC2.

Methods: We randomly divided 40 male SD rats into four groups. In the brachial plexus avulsion group, C8-T1 roots were avulsed from the spinal cord at the lower trunk level. In the K252a group, 5uL K252a was applied intrathecally daily for three days after avulsion. In the sham surgery group, expose only and without damage. The control group did not undergo any treatment. Mechanical hyperalgesia and cold allodynia were analyzed by electronic pain measuring instrument and acetone spray method at different time points on days 1, 3, 7, 10, 14, and 21 after surgery. At 21 days after surgery, the expression of BDNF and TrkB in dorsal horn neurons and GFAP in astrocytes were detected by immunohistochemistry at the C5-T1 segment of the spinal cord. The expression levels of BDNF, TrkB, and KCC2 in the C5-T1 spinal cord were measured by Western Blot at 7 and 21 days.

Results: Mechanical hyperalgesia and cold allodynia were significantly reduced in the K252a group compared with the brachial plexus avulsion group. Compared with the BPA group, BDNF, TrkB and GFAP were significantly decreased in the K252a group at 21 days after treatment by immunohistochemical test. In the WB test, the expressions of BDNF and TrkB in the K252a group were quantitatively detected to be decreased, while the expression of KCC2 was increased, which was obvious at 7 and 21 days.

Conclusion: BDNF-TrkB-KCC2 pathway can significantly relieve neuropathic pain after BPA, and is a potential target for the treatment of neuropathic pain.

Keywords: BDNF; K252a; KCC2; TrkB; brachial plexus avulsion; neuropathic pain.

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Conflict of interest statement

The authors declare that there is no conflict of interest.

Figures

FIGURE 1
FIGURE 1
The brachial plexus of rat
FIGURE 2
FIGURE 2
The lower trunk of brachial plexus avulsion in surgery. The black arrow shows the dorsal root ganglion
FIGURE 3
FIGURE 3
The hind paw‐withdrawal threshold of the operative side showed no difference at 0 days before operation among all groups and significantly decreased at 1, 3, 7, 10, 14, and 21 days after operation in the BPA group (p < .05). There was no difference at all time points between the control group and the sham operation group. After treatment with K252A, the hind paw‐withdrawal threshold was significantly higher at all postoperatively time points and consistently until 21 days postoperatively. * p < .05
FIGURE 4
FIGURE 4
At 0 days before the operation, there was no difference in the cold allodynia scores among all group. At all time points after the operation, the score of cold allodynia in the BPA group was significantly increased, which was significantly different from that in the control group and the sham operation group. After treatment with K252a, the score of cold allodynia was significantly decreased at 1, 7, 10, and 21 days postoperatively and had no difference compared with the control group and the sham operation group. The score of cold allodynia was not significantly decreased at 3 and 14 days after treatment with K252a and had no difference compared with that of the BPA group, control group, and sham operation group. * p < .05
FIGURE 5
FIGURE 5
The expression of BDNF, GFAP, TrkB in the spinal cord. (a) The expression of BDNF was significantly increased in the BPA group and decreased to normal level after treatment with K252a. (b) The expression of GFAP in the spinal cord was significantly increased in the BPA group and decreased to normal level after treatment with K252a. (c) The expression of TrkB was significantly increased in the BPA group and decreased to normal level after treatment with K252a. ** p < .01
FIGURE 6
FIGURE 6
The expression of BDNF, GFAP, and TrkB at the C5–T1 dorsal horn segment in the four groups 7 days after surgery was increased compared with the sham operation and control group, but significantly decreased with K252a treatment compared with the sham operation and control group. Scale bars represent 20 µm
FIGURE 7
FIGURE 7
The expression of BDNF, GFAP, TrkB in the spinal cord at 21 days. (a) The expression of BDNF was significantly increased in the BPA group and decreased to normal level after treatment with K252a. (b) The expression of GFAP in the spinal cord was significantly increased in the BPA group and decreased to normal level after treatment with K252a. (c) The expression of TrkB was significantly increased in the BPA group and decreased to normal level after treatment with K252a. ** p < .01
FIGURE 8
FIGURE 8
The expression of BDNF, GFAP, and TrkB at the C5–T1 dorsal horn segment in the four groups 21 days after surgery was increased compared with the sham operation and control group, but significantly decreased with K252a treatment compared with the sham operation and control group. Scale bars represent 20 µm
FIGURE 9
FIGURE 9
Changes in expression of BDNF, KCC2, TrkB in the spinal cord after brachial plexus avulsion and K252a treatment. (a, c, e) There was no difference in BDNF, TrkB, and KCC2 expression between the control group and the sham operation group at 7 days. The expressions of BDNF and TrkB in the BPA group increased significantly at 7 days after surgery, compared with those in the control group and the sham group, and decreased significantly at 7 days after treatment with K252a. The expression of KCC2 decreased significantly in the BPA group at 7 days, and increased significantly in the BPA group at 7 days, and increased significantly after treatment with K252a. (b, d, f) Twenty‐one days after surgery, BDNF and TrkB expression increased significantly after BPA, but decreased significantly after K252a treatment, while KCC2 expression decreased significantly after BPA and increased significantly after K252a treatment. (g) Western blot analysis of BDNF, TrkB, and KCC2 in four groups at 7 and 21 days, respectively. ** p < .01
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